You think the hairy root culture would be impractical?

I don't, not even a little because its exploiting the one part of the plant that produce the safrole and basicly turns a large vessle into a solid block of rootbark. Its simular to having an algee produce safrole for you from Eugenol. The material would already be finely divided and it would be a simple passage of steam into the tank to yeild the goods. Transfer a portion of this to a new container to culture before killing the old with steam and we have a very viable rapidly sustainable source of safrole.

IIRC the "Special bacteria" can be harvested with ease from.... dirt. Once you got that the rest should be easy it seems.

Why is that not easy? As much as I would love to tackle this last thing I need right now is another project but I think you folks may be missing the bigger picture here. Safrole is one thing but what about other substances produced in roots? DMT is the first to come to mind. And im pretty confident that it can be done in equipment only a little bit removed from brewing setups. Would you not like a setup that you mearly feed with say tryptophan and it produces gallons of DMT laced liquid?

This warrents its own thread but Ill leave you with a couple tidbits to think about.

PnP-24 (Postdoctoral)
A cytochroMe P450 enZyMe froM illiciUM pArViFlorUM cAPAble of
cAtAlyZing the forMAtion of A MethylenedioXy bridge on eugenol to Produce SAfrole

Phenylpropanoids constitute a major group of secondary compounds present in the plant kingdom.  The
volatile phenylpropenes, a subgroup of the phenylpropanoids in which the propyl side-chain is reduced,
include the well-known spice eugenol as well as safrole, myristicin, apiol, and dillapiole; they are also believed
to function as defense toxins against herbivores and pathogens as well as in attracting pollinators.  Such
compounds have been found in basil, parsley, dill, yellow anisetree and many other plant species dispersed
throughout angiosperm taxa, although in many cases closely related species do not make the same set of
phenylpropene compounds, at least not in detectable levels.  Here we report a putative safrole synthase
gene, isolated from  Illicium parviforum  (yellow anisetree), encoding a cytochrome P450 enzyme that is
capable of converting eugenol to safrole via the formation of a methylendioxy bridge.  High levels of safrole
are detected in both leaves and fowers of I. parviforum and lower levels are present in the stem.  Transgenic
yeast cell cultures and Arabidopsis plants overexpressing the safrole synthase gene and fed with eugenol
are able to convert it to safrole.  In vitro enzyme assay with a microsomal fraction of Arabidopsis plantlets
shows detectable activity.  In addition, while petunia fowers normally emit high levels of isoeugenol and very
low levels of eugenol, but no safrole or isosafrole, transgenic petunia fowers transformed with the safrole
synthase gene emit low levels of isosafrole in addition to isoeugenol and eugenol

Link:http://www.psna-online.org/newsletters/PSNAN461web.pdf

You can see in this link that they successfully have modified yeast to produce Safrole from Eugenol. HRCs will act in a simular manner allowing the P450 enzyme to be active in the biosynthesis of Safrole.

I for one do not see any of this to be hard but I guess to each his own. Hard is figuring out how to synthesis Safrole from Eugenol and go thru Multisteps and low yeilds to get to what a little bit of for thought wil get you  with ease.

And your are wrong again.


 Genetic modification while it can be complicated does not have to be. There are many lowtech variations to acheive gene isolation and expression in a culture but thats for another place and time.
Still I only included that as a source to show that these modifications will produce a biofactory in a dish to convert Eugenol to Safrole. These HRCs seem to have yall a little off base as to what they are. You do NO! modification of the root but the DNA splicing is done thru nothing more then infecting it with a common bacteria that can be isolated from soil. Once its infected the bacteria carrys its own DNA into the root which will cause roots to grow wildly hence the term Hairy root culture.


There is a Rosebush that a guy went a bit further and modified the bacteria with DNA from a jellyfish and still 4 years later it Glows showing that once the dirtywork is done you will have a constant supply of Safrole cheep and easy. Thats complicated but simply infecting a sassafrass tree with a bacteria and havesting the root to keep alive in a reactor does not seem like to big a project for someone to pull off at home.

I don't know,,, like I said I think everyone is reading to much into this and picturing gene guns lab coats and sterile rooms when its really just an infection and brewing equipment.






Aside from all this fearfulness everyone do yourself a favor if you have a large cheep supply of Eugenol then feed it to your Sassafrass plant slowly right before harvest. The CP450 enzyme will be active and able to convert any Eugenol to safrole increasing yeilds.

Im going to start a thread on the subject soon enough where we could continue the discussion. I have no personal experiance but from everything I have read it does not seem that large of a task other then the isolation of the bacteria which will involve abit of petri dish cultures and identification/isolation of the correct bacteria but after that the rest does not seem to hard to pull off.

Legal is where my mind has been heading with the thought of it but lets face it, legal don't always grab the attention of folks around these parts of the woods .

I believe simular things can be done to produce ephedrine or opium but that would take a slightly different aproach due to the area of active enzymes in the plant. Sandlewood for instance is produced in the bark of the tree which would require a culture of the bark to be sustained by itself which im not so sure is feesaible but roots being the base of a plant are able to provide themselfs with there own food without the need of something to transport that energy from elseware. Leave cultures should be possible as well with relative ease though.

If someone was to want to do something legal though there is much more potential then sandlewood as far as profit is concerned in the medical field in producing precursors for various medicines required in the field. The HRCs can single handedly produce total synthesis of complex organic molecules otherwise not economic for the medical field to produce on its own. Think of the possiblility this would have if you could have it produce something like Taxol for instance. The possiblitys are endless and tech new enough that someone with the right determination and motivation could stand to profit lovely from there venture by leading the field in most areas they decided to corner.

Look at this short list and imagine the possibilitys this hs in the medical field for someone thats not even attempting any major gene alteration but only using what the plants provide in the first place.
http://www.rain-tree.com/plantdrugs.htm

i love this place--hairy root cultures that glow in the fuckin dark

--too cool for words where these ideas are headed--keep up the AWESOME investigations

They have also done some work with making plants glow -- google glowing tobacco.. that ought to give you some info on it. IIRC biology-online at some point had some pretty good stuff on it.. but this is just a vague memory.

This is a really cool thread and wanted to throw my meager 2 cents worth in.  Last Fall I went on a sabbatical trip to the Smokies and while I was there I researched sassafras - got a plant guide, learned what it looked like etc.  The harvesting advice I got was to pull up the young trees as well as digging up root bark.  I didn't collect a whole lot, small bag full - more for the fun of it than any particularly nefarious purpose.  I figured I'd distill off what I could for the experience and use it to flavor my herbal extracts.

I finally got around to working with it this weekend.  What I noticed was the saplings I pulled up had a streak of grayish stuff near the roots and joints.  This stuff smells strongly of sassafras.  I got a suspicion as I was smelling and working with the stuff that the safrole is in the sap which I did confirm (at least via the folk knowledge).  The leaves smell lemony which helps identify the plant - its kind of funky when it's young having several different shaped leaves.  So when I was processing the stems I smelled and cut my way through the scraps.  The limonene and pinene smell was strong in some stems that didn't have the darkish streaks (sap) in them so I didn't put those in my column.

Regarding the processing of Hoya Santa I would suggest setting up some drying trays over mol sieve or silica gel - once the leaves are dry then you can grind them up into a powder.   (that stuff grows wild down here too - is on my list of stuff to find - it's a wonder I don't kill myself looking at stuff when I'm driving)